The present invention relates to a precious metal-recombinant apoferritin complex produced with a gene recombination technique and a method for producing the same, and techniques related thereto.
In recent years, in-depth research on bioelectronics, which is a combination of biotechnology and electronics, has been conducted, and some products such as biosensors employing proteins such as enzymes already have been put to practical use.
As one attempt to apply biotechnology to other fields, there is research in which fine particles made of metal or metal compounds are incorporated into apoferritin, which is a protein having the function of holding metal compounds, to produce the fine particles having uniform sizes of nm order. Research to introduce various metals or metal compounds suitable to the application of the fine particles into apoferritin has been under way.
Hereinafter, apoferritin will be described. Apoferritin is a protein that exists widely in the biological world and has the role of regulating the amount of iron, which is an essential trace element in living organisms. A complex of iron or an iron compound of apoferritin is called ferritin. If iron is present in an amount more than necessary, it is harmful to living organisms, so that excessive iron is stored in the form of ferritin. The ferritin releases an iron ion as necessary and is converted back to apoferritin.
FIG. 1 is a schematic view showing the structure of ferritin (iron-apoferritin complex). As shown in FIG. 1, ferritin is a spherical protein having a molecular weight of about 460,000 in which 24 monomer subunits constituting one polypeptide chain are assembled by non-covalent bonds, has a diameter of about 12 nm, and exhibits higher thermal stability and higher pH stability than those of common proteins. A hollow holding portion 4 having a diameter of about 6 nm is present in the center of this spherical protein (outer shell 2), and the holding portion 4 is connected to the outside via a channel 3. For example, when incorporating a bivalent iron ion into ferritin, the iron ion enters it through the channel 3 and is oxidized in a site called “ferrooxidase center” in a subunit in a portion thereof, and then reaches the holding portion 4 and is concentrated in a negative load region on the inner surface of the holding portion 4. Then, 3000 to 4000 iron atoms assemble and are held in the holding portion 4 in the form of ferrihydrite (5Fe2O3.9H2O) crystal.
In this specification, a fine particle including a metal atom held in the holding portion is referred to as a “core”. The diameter of the core 1 shown in FIG. 1 is substantially equal to the diameter of the holding portion 4, which is about 6 nm.
The core 1 can be removed by a comparatively simple chemical operation, and the particle constituted only by the outer shell 2 without the core 1 is called apoferritin. Using apoferritin, an apoferritin-fine particle complex in which a metal or a metal compound other than iron is supported artificially has been produced.
To date, it has been reported that metals such as manganese (P. Mackle, 1993, J. Amer. Chem. Soc. 115,8471–8472; F. C. Meldrum et al., 1995, J. Inorg. Biochem. 58, 59–68), uranium (J. F. Hainfeld, 1992, Proc. Natl. Acad. Sci. USA 89,11064–11068), beryllium (D. J. Price, 1983, J. Biol. Chem. 258, 10873–10880), aluminum (J. Fleming, 1987, Proc. Natl. Acad. Sci. USA, 84, 7866–7870), zinc (D. Price and J. G. Joshi, Proc. Natl. Acad. Sci. USA, 1982, 79, 3116–3119), and cobalt (T. Douglas and V T. Stark, Inorg. Chem., 39, 2000, 1828–1830) or metal compounds are introduced into apoferritin. The diameter of the core 1 made of these metals or metal compounds is also substantially equal to the diameter of the holding portion 4 of the apoferritin, which is about 6 nm.
The process for forming the core 1 including an iron atom in ferritin in the natural world proceeds generally in the following manner.
An amino acid having a negative charge at pH 7–8 is exposed onto the surface of the channel 3 (see FIG. 1) for connecting the outside and the inside of the ferritin particle, and a Fe2+ ion having a positive charge is captured by the channel 3 by electrostatic interaction. The channels 3 are present in the number of 8 per apoferritin.
As on the inner surface of the channel 3, a large number of glutamic acid residues, which are amino acid residues having a negative charge at pH 7–8, are exposed onto the inner surface of the holding portion 4 of the ferritin, and Fe2+ ions captured from the channel 3 are oxidized at the ferroxidase center and led to further inside of the holding portion 4. Then, the iron ions are concentrated by electrostatic interaction and nucleus formation of a ferrihydrite (5Fe2O3.9H2O) crystal occurs.
Thereafter, iron ions that are sequentially captured are attached to the nucleus of this crystal, so that the nucleus made of iron oxide is grown and thus the core 1 having a diameter of 6 nm is formed in the holding portion 4. The capture of iron ions and the formation of the nucleus made of iron oxide are performed generally in the manner as described above.
Next, an operation for introducing iron to apoferritin will be described below.
First, a HEPES (2-[4-(2-hydroxyetyl-1-piperazinyl)]-ethanesulfonic acid) buffer solution, an apoferritin solution, and an ammonium iron sulfate (Fe(NH4)2(SO4)2) solution are mixed in this order to prepare a ferritin solution. In this ferritin solution, the final concentrations of the HEPES buffer solution, apoferritin and ammonium iron sulfate are 100 mmol/L (pH 7.0), 0.5 mg/mL, and 5 mmol/L, respectively. All the operations for preparing ferritin are performed at room temperature and stirring is performed with a stirrer.
Next, in order to complete a reaction for capturing iron ions into apoferritin and an oxidation reaction of the captured irons, the ferritin solution is allowed to stand over night. This operation introduces iron oxides having uniform sizes into the holding portion of apoferritin, so that ferritin (a complex of apoferritin and a fine particle) is produced.
Next, the ferritin solution is placed in a container, and centrifuged at 3,000 rpm with a centrifugal separator for 15 to 30 min to remove a precipitate. Then, the resultant supernatant obtained after the precipitate is removed is centrifuged further at 10,000 rpm for 30 min so as to precipitate an unwanted ferritin aggregate and remove it. At this point, ferritin is present in the supernatant in the form of a dispersion.
Next, as the solvent of this supernatant, the 100 mmol/L HEPES buffer solution of pH 7.0 is replaced by a 150 mmol/L NaCl solution by dialysis to prepare a new ferritin solution. Here, the pH does not necessarily have to be adjusted.
Then, this ferritin solution is concentrated to an arbitrary concentration between 1 and 10 mg/mL, and then CdSO4 is added to this solution such that the final concentration thereof becomes 10 mmol/L to aggregate the ferritin.
Next, the ferritin solution is centrifuged at 3,000 rpm for 20 min to precipitate a ferritin aggregate in the solution. Thereafter, the buffer component in the solution is replaced by a 10–50 mmol/L Tris buffer solution of pH 8.0 containing 150 mmol/L NaCl by dialysis.
Next, the ferritin solution is concentrated and then is filtrated by gel filtration column to remove an aggregate of ferritin particles, so that discrete ferritin including iron oxide can be obtained.
The mechanism for capturing iron ions into ferritin and a method for preparing ferritin including iron oxide have been described above. Since all the other metal ions that have been reported so far to be introduced are positive ions, it is believed that the capture of these metal ions to apoferritin substantially in the same mechanism as in the case of iron ions. Therefore, the other ions basically can be introduced into apoferritin substantially in the same operations as in the case of iron ions.
Regarding apoferritin, the size of a particle that can be held slightly varies with the type of the organism from which it is derived. Furthermore, there are spherical proteins that have similar structures to that of apoferritin and can hold inorganic particles inside. Examples thereof include Listeria ferritin derived from Listeria monocytogenes and Dps protein. There are proteins that are not spherical but can hold an inorganic particle similarly to ferritin, such as outer shell proteins of virus such as CCMV.
In the specification of the present application, proteins that can hold inorganic particles inside such as spherical proteins, outer shell proteins of virus are referred to as “cage-like proteins”.
These cage-like proteins can hold inorganic particles including iron.
Thus, ferritin holding a metal ion such as iron can be produced in the above-described method. However, since the inner surface of the channel 3 of apoferritin and ferritin is positively charged as a whole, it is difficult to capture ions having the same negative charge into apoferritin.
On the other hand, gold, platinum or the like cannot be ionized alone in an aqueous solution, and only can be present as complex ions in an aqueous solution. Therefore, they are often used in the form of negative ions of chloroauric acid ions (AuCl4)− or (PtCl4)2−. Consequently, it was difficult to capture precious metal atoms such as gold or platinum into apoferritin in the prior art.